The synthesis of saturated fatty acids is catalyzed by an enzyme system in the cytosol portion of cells. Reducing equivalents required for this reaction are supplied by the malic enzyme and pentose shunt dehydrogenases. Components of the dissociable Escherichia coli fatty acid synthetase (including acyl carrier protein) have been separated, and intermediate steps have been clarified. The fatty acid synthesizing systems of yeast and animal cells catalyze a series of reactions analogous to that in E. coli; however, the component activities reside in a high molecular weight enzyme complex. Malic enzyme has been isolated from pigeon liver in crystalline form and extensively characterized. Recent studies in our laboratory have shown that the activity of this enzyme is regulated by substrate inhibition at high (but physiological) levels of malate, and that it exhibits "Half-of-the-Sites" reactivity, possibly due to negative cooperativity between identical subunits. The ultimate objective of this project is to elucidate the mechanism and regulation of fatty acid synthesis in animals. For this purpose, detailed kinetic, binding, and structural studies will be carried out on malic enzyme to delineate the molecular basis of cooperativity. These results will allow us to evaluate the role of subunit interactions in catalysis, and in the modulation of malic enzyme activity by a mechanism of autonomous control. "Half-of-the-Sites" reactivity has been shown for the fatty acid synthetase of yeast. In our studies, chemical modification and substrate inhibition experiments will be performed on the chicken liver complex to uncover such behavior, and its possible significance in the physiological regulation of fatty acid synthetase activity. Ultimately, results of these studies will enhance our knowledge of, and help us to resolve the cellular machinery for lipogenesis in animals.